Use of fluorescence for the quick and easy determination of s-adenosylmethionine, s-adenosylhomocysteine and homocysteine

ABSTRACT

The invention provides immunochromatographic test strips and methods for detecting and quantifying S-Adenosylmethionine (SAM), S-Adenosylhomocysteine (SAH) and Homocysteine (HCy) in a sample, comprising: (a) making fluorophore conjugated antibodies; (b) immobilizing SAM, SAH and HCy on a solid support; (c) providing a sample, combining said sample with a conjugate selected from the group consisting of lanthanide chelate conjugates and quantum dot conjugates (QD) with anti-SAM, anti-SAH or anti-HCy, wherein said combining is performed under conditions that allow formation of a competitive complex comprising said conjugate, said SAM, SAH or HCy on the solid support and SAM, SAH or HCy in a sample when present; and (d) detecting the presence of the complex, if present, by monitoring a spectral emission mediated by the fluorescent conjugates in the complex, wherein the emission indicates the presence and quantity of SAM, SAH or HCy in the sample.

This application claims the priority benefit under 35 U.S.C. section 119of U.S. provisional Patent Application No. 62/166,044 entitled “Use OfImmunological And Chemical Methods For The Quick And Easy DeterminationOf SAM, S-Adenosylhomocysteine And Homocysteine” filed on May 25, 2015,and which is in its entirety herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the use of fluorescent materials suchas quantum dots, fluorescent lanthanide metal chelate complexes, andcolloidal microspheres in the immunological determination ofS-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and C-reactionprotein (CRP). The invention further relates to the use of aphotochemical method for the determination of homocysteine (HCy) in adry strip and the combinations of both methods. The invention furtherrelates to the quantitative measurement of SAM, SAH and HCysimultaneously using fluorescence-optical density devices that readimmunological fluorescence and photochemical colors simultaneously forquick and convenient reporting. The present invention further relates toassays of clinical samples.

This invention also relates to fluorescent compounds useful as indicatormolecules for detecting the presence or concentration of an analyte in amedium, such as a liquid, and to methods for achieving such detection.More particularly, the invention relates to fluorescent lanthanide metalchelate complexes and their use as indicator molecules for detecting thepresence or concentration of an analyte such as SAM, SAH and CRP in amedium, including a liquid medium such as a biological fluid or otherbiological samples.

The invention additionally relates to the development of an assay systemcapable of discriminating mixtures of cardiovascular risk factoranalytes for the prediction of coronary heart disease and stroke. Theinvention is also directed to the determination of SAM and SAH, HCy andCRP to determine cardiac care and cardiac prognosis. The instantinvention is also particularly useful in the field of in vitro diagnosis(IVD) and point-of-care testing (POCT).

BACKGROUND OF THE INVENTION

In biology it is of interest to mark structures such as cells or viruseswith fluorescent materials for accurate identification, ease ofdetection and microscopic analysis. Traditionally, organic dyefluorophores have been the favored materials and have the capability tobe modified with a range of materials, enabling targeted binding to awide range of biological structures based on known affinities andchemistries. Upon binding of the dye to the target biological material,an activating light of a given wavelength is used to excite the dye,from which it responds by fluorescently emitting a characteristic lightradiation specific to the properties of the organic dye employed.However, traditional organic dyes have numerous limitations when used totag biological materials.

Semiconductor fluorescent nanocrystals (“quantum dots”) are nanometersized semiconductor, light-emitting crystals, spherical in shape andhave superior fluorescent properties to organic dyes. Quantum dots aregenerally synthesized with Type II-VI (e.g. CdSe, CdTe, CdS and ZnSe) orType III-V (e.g. InP and InAs) column elements from the periodic tableand can be capped with numerous shells, layers or molecules to modifytheir physical properties, such as for surface functionalization.Integration of quantum dots in biology was achieved in breakthroughsshowing that highly luminescent quantum dots could be made water-solubleand subsequently biocompatible using surface modification techniquessuch as silica/siloxane coatings or direct absorbtion of bifunctionalligands, which presented them useful tools in biology. Quantum dots areemerging as the new biological label with applications and propertiessuperior to traditional fluorescent proteins and organic dyes.

Most of the limitations with traditional organic dyes are a result ofthe extremely limited absorptive and emissive capabilities. The firstshortcoming is that the peak emission of organic dyes cannot bealtered—each dye corresponds to a different molecule with a differentpre-set emission wavelength, or fluorescent color, that is set bynature. The second shortcoming is the narrow absorption pattern oforganic dyes—dyes tend to display absorption peaks that are not alwaysin convenient regions of the spectrum, making the excitation of variousorganic dyes challenging and costly. The third shortcoming is that ofuneven absorption and emission peaks—organic dyes have a tendency toproduce “shoulders” in the geometry of their emission and absorptionpeaks, which is a major disadvantage in applications that requireGaussian type emission patterns to work correctly. An additionalshortcoming is that of stability—the lifetime of organic dyes varies butis generally low relative to that of other tagging mechanisms andorganic dye fluorescence is controlled entirely by the molecular bondingproperties of each individual dye. Finally, incident radiation absorbedby an organic dye molecule moves electrons into excited states,whereupon they decay and release light radiation. This emission cannotbe altered because it corresponds to pre-set excited states of the dyemolecule that are inherent to every molecule of that type.

Whereas the light emission ranges and possible forms of organic dyes arevery limited, quantum dots can be made to emit light at any wavelengthin the visible and infrared ranges, and can be inserted almost anywhere,including in liquid solutions, dyes, paints, epoxies, and sol-gels.Furthermore, quantum dots can be attached to a variety of surfaceligands, and inserted into a variety of organisms in vivo or in vitro.

Numerous methods exist for covalently linking biological molecules toquantum dots to create a bio-molecular conjugates (“bioconjugate”) orfunctional quantum dot which are used in labeling, detection and imagingapplications to attach or bind a quantum dot to a biological materialbased on specific chemical or biological affinity. These methods employa variety of chemistries to water-soluble quantum dots from whichseveral cross-linker molecules can be coupled to enable the attachmentof the primary functional biomaterial. Other examples of bioconjugatetechniques enabling the attachment of various materials to quantum dotsare known to those skilled in the art.

Generally, bioconjugation methods are classified into mechanisms using:(1) Biofunctional linkages, (2) Electrostatic attraction, (3)Hydrophobic attraction, (4) Silanization, and (5) Nanobead linkages.Examples of methods employing bioconjugative techniques arepolyethylglycol modification of the underlying carboxyl quantum dots,and optimization of the surface loading of amino groups for highconjugation efficiency and specificity. Another example is modifying thequantum dots with peptides through the amino or carboxyl groups at theterminus, or using other residues, small molecules, proteins, or nucleicacids, and other methods known to those skilled in the art. Morespecifically, schemes used for the conjugation of antibodies to quantumdots are based on well-known chemistries using the fast and efficientcoupling of thiols to maleimide groups, with reactive groups such asprimary amines, alcohols, carboxylic acids and thiols used to link theantibodies to the quantum dots.

Quantum dots represent a marked increase in performance over standardorganic dyes, because they can be tuned to absorb or emit at any visibleor infrared wavelength and can be fabricated into a great variety offorms and media, eliminating completely the shortcomings of dyes. Theseunique abilities are due to their very small sizes (typically between1-10 nm in diameter). The small size and its direct relationship tofluorescence also allows for incredible versatility and flexibility ofform, letting phosphors match whatever shape their underlyinglight-emitting diode (LED) needs to assume.

When light impinges on quantum dots, it encounters discretized energybands specific to the quantum dot. The discretized nature of quantum dotbands means that the energy separation between the valence andconduction bands (the bandgap) can be altered with the addition or thesubtraction of just one atom—making for a size dependent bandgap.Pre-determining the size of the quantum dots fixes the emitted photonwavelength at the appropriate customer-specified color, even if it isnot naturally occurring—an ability limited only of quantum dots.

Additionally, it is also known that certain rare-earth metal chelatesemit visible light upon irradiation with UV light and different forms ofvisible light (e.g., violet or blue light), an emission which ischaracterized by the chelated cation. Some lanthanide ions, such asthose of europium (Eu³⁺), Samarium (Sm³⁺), terbium (Tb³⁺), and to alesser extent dysprosium (Dy³⁺) and neodymium (Nd³⁺), exhibit typicalfluorescence characterized by the ion, especially when chelated tosuitable excitation energy mediating organic ligands. The fluorescentproperties of these compounds—long Stokes' shift, narrow band-typeemission lines, and unusually long fluorescence lifetimes—have made themattractive candidates for fluorescent immunoassays and time-resolvedfluorometric techniques.

The major emission lines of these fluorescent lanthanide chelates areformed from a transition called hypersensitive transition and are around613-615 nm with Eu³⁺, 545 (and 490) nm with Tb³⁺, 590 and 643 nm withSm³⁺, and 573 with Dy³⁺. Radiation is typically absorbed by the chelatesat a wavelength characteristic of the organic ligand and emitted as aline spectrum characteristic of the metal ion because of anintramolecular energy transfer from the ligand to the central metal ion.The organic ligand absorbs energy and is raised or excited from itssinglet ground state, S₀, to any one of the vibrational multiplets ofthe first singlet excited state, S₁, where it rapidly loses its excessvibrational energy. At this point, there are two possibilities:relaxation by an S₁→S₀ transition (ligand fluorescence) or intersystemcrossing to one of the triplet states, T₁.

Fluorescent europium chelates are known to exhibit large Stokes shifts(˜290 nm) with no overlap between the excitation and emission spectraand very narrow (10-nm bandwidth) emission spectra at 615 nm. Inaddition, the long fluorescence lifetimes (measurable in microsecondsinstead of the nanosecond lifetimes measurable for conventionalfluorophores) of the chelates help filter out noise and otherinterference having a low fluorescent lifetime. The long fluorescentlifetimes thus permit use of the chelates for microsecond time-resolvedfluorescence measurements, which further reduce the observed backgroundsignals. Additional advantages of using europium chelates include thateuropium chelates are not quenched by oxygen.

In specific binding assays, sensitivity is of prime importance due tothe generally low analyte levels that are measured. Radioimmunoassaysensitivity limits the assay to measurements of concentration of 10⁻¹²M, and more often only in the 10⁻⁸ to 10⁻¹⁰ M range. In addition,radiolabels suffer from the drawbacks of short half life and handlinghazards.

In fluorescence spectroscopy assays, a sample containing a fluorescentspecies to be analyzed is irradiated with light of known spectraldistribution within the excitation spectrum of the target fluorescentspecies. The intensity of the resulting characteristic emission spectrumof the fluorescent target molecules is determined and is related to thenumber of target molecules.

The sensitivity of fluorescence assays, although theoretically veryhigh, is limited by the presence of background fluorescence. Backgroundsignal levels are picked up from competing fluorescent substances, notonly in the sample, but also in materials containing the sample. This isan especially serious problem in quantitative measurements of speciesassociated with samples containing low concentrations of desired targetfluorescent molecules such as found in biological fluids. In manysituations, it is impossible to reduce the background sufficiently (byappropriate filtration and other techniques known in the art) to obtainthe desired sensitivity.

Time resolution offers an independent means of isolating the specificfluorescent signal of interest from nonspecific background fluorescence.Time resolution is possible if the label has much longer-livedfluorescence than the background, and if the system is illuminated by anintermittent light source such that the long-lived label is measurableduring the dark period subsequent to the decay of the short-livedbackground.

Certain fluorescent molecules have been commonly used as tags fordetecting an analyte of interest. Organic fluorescent dyes are typicallyused in this context. However, there are chemical and physicallimitations to the use of such dyes. One of these limitations is thevariation of excitation wavelengths of different colored dyes. As aresult, the simultaneous use of two or more fluorescent tags withdifferent excitation wavelengths requires multiple excitation lightsources.

A drawback of organic dyes is the deterioration of fluorescenceintensity upon prolonged and/or repeated exposure to excitation light.This fading, called photobleaching, is dependent on the intensity of theexcitation light and the duration of the illumination. In addition,conversion of the dye into a nonfluorescent species is irreversible.Furthermore, the degradation products of dyes are organic compoundswhich may interfere with the biological processes being examined.

Additionally, spectral overlap exists from one dye to another. This isdue, in part, to the relatively wide emission spectra of organic dyesand the overlap of the spectra near the tailing region. Few lowmolecular weight dyes have a combination of a large Stokes shift, whichis defined as the separation of the absorption and emission maxima, andhigh fluorescence output. In addition, low molecular weight dyes may beimpractical for some applications because they do not provide a brightenough fluorescent signal.

Furthermore, the differences in the chemical properties of standardorganic fluorescent dyes make multiple, parallel assays impractical asdifferent chemical reactions may be involved for each dye used in thevariety of applications of fluorescent labels.

Thus, there is a continuing need in the assay art for labels with thefollowing features: (i) high fluorescent intensity (for detection insmall quantities), (ii) adequate separation between the absorption andemission frequencies, (iii) good solubility, (iv) ability to be readilylinked to other molecules, (v) stability towards harsh conditions andhigh temperatures, (vi) a symmetric, nearly gaussian emission lineshapefor easy deconvolution of multiple colors, and (vii) compatibility withautomated analysis. At present, none of the conventional fluorescentlabels satisfies all of these requirements.

While fluorescent emissions from functional quantum dot bioconjugateshave been used to detect the presence or absence of a target substratein a sample, at present there remains no fast and effective method andapparatus for measuring SAM and SAH.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates two embodiments of the lateral flowimmunochromatographic test strips of the invention.

FIG. 2 shows the standard curve for the SAM fluorescentimmunochromatographic test strip of example 1 invention.

FIG. 3 shows the standard curve for the SAH fluorescentimmunochromatographic test strip of example 2.

FIG. 4 shows the standard curve for a CRP fluorescentimmunochromatographic strip of example 4.

FIG. 5 illustrates the flow cytometry (FCM) results from cells doublestained with Alexa Fluor 647 conjugated anti-SAM 118-6 antibody derivedfrom hybridoma clone 118-6 (Cat# MAF00201, Arthus Biosystems, VA) at 4.5μg/ml. A sample of the subject hybridoma clone 118-6 was deposited atthe China Center For Type Culture Collection (CCTCC) Address: WuhanUniversity, Wuhan Zip code: 430072 on Sep. 16, 2017, and has beenassigned the CCTCC number C2017179.

FIG. 6 shows FCM results from cells double stained with Alexa Fluor 488conjugated anti-SAH antibody 301-3 (Cat #MAF00301, Arthus Biosystems,VA) at 45 μg/ml.

FIG. 7 illustrates the Laser Scan Confocal Microscopy (LSCM) results ofL02 and HepG2 cells that were cultured for 40 h and then stained withthe SAMe fluorescence labelled anti-SAM and anti-SAH antibodies of theinvention.

FIG. 8 shows simple diagrams illustrating how the two formats of TR-FRETtechnology may be used to quantitatively measure SAM and SAH using thebio-conjugates described in this invention.

SUMMARY OF THE INVENTION

The present invention provides quantum dots having attached thereto anantibody selected from the group consisting of anti-SAM, anti-SAH,anti-HCy and anti-CRP antibodies.

The invention also provides an immunochromatographic strip havingincorporated therein quantum dots covalently bonded to anti-SAM,anti-SAH, anti-HCy and anti-CRP antibodies.

The present invention is also directed to the use of quantum dots basedimmunoassays in combination with chemical methods to measure threeclosely related bio-molecules in a metabolic pathway simultaneously.

The invention is also a method of determining risk of experiencing amajor adverse cardiac event in a patient, within one year frompresentation of at least one symptom of acute coronary syndromecomprising the steps of: (a) obtaining a test sample from said patient;(b) determining the amount of at SAM, SAH, HCy and optionally C reactiveprotein using a quantum dot based assay; (c) calculating the MI in saidtest sample; and c) comparing the amount of said four biomarkers tobiomarker reference standards, wherein said risk is determined byresults of said comparison.

The invention further provides a method for assaying homocysteine in asample, said method comprising the steps of: (i) contacting said samplewith a homocysteine-converting enzyme that produces SAH and (ii) thenmeasuring SAH using an immunochromatographic strip as described above.

The invention also provides a lateral flow immunoassay test strip fordetecting and quantifying the presence of SAM, and SAH alone orsimultaneously in a fluid sample, comprising a membrane strip coatedwith a SAM or SAH-protein conjugate on a test line, and particlesconjugated with their antibodies respectively.

The invention is also directed to a fluorescent lanthanide chelateconjugated to an antibody selected from the group consisting ofanti-SAM, anti-SAH, and anti-CRP antibodies and use of the conjugates inmaking immunochromatographic strips.

The invention further provides a method of detecting and quantifying SAMand SAH in a sample, comprising: (a) providing a sample containing orsuspected of containing SAM and SAH on a solid support; (b) combiningsaid sample with a semiconductor nanocrystal anti SAM antibody and antiSAH antibody conjugate, wherein said combining is performed underconditions that allow formation of a complex comprising said conjugateand said SAM and SAH, when present; (c) removing any unbound conjugate;and (d) detecting the presence of the complex, if present, by monitoringa spectral emission mediated by the semiconductor nanocrystal in thecomplex, wherein the emission indicates the presence and quantity of SAMand SAH in the sample.

The invention further relates to the use of a SAM immuno-chromatographicstrip to determine and monitor the levels of SAM in patients afflictedwith a disease selected from the group consisting of depression,osteoarthritis, liver and gall bladder diseases and then proposing atherapeutic regimes for administering S-Adenosyl-methionine.

The invention also provides a method for determining the effectivenessof a diet program for administration to a patient having at least onediet-responsive condition comprising the steps of: (a) selecting aplurality of patients, each having at least one diet-responsivecondition; (b) identifying in said patient the body mass index and atleast one other quantifiable indicator selected from methylation indexand SAM levels for each of said diet-responsive conditions and measuringsaid at least one indicator for each of said patients during a baselineperiod; (c) monitoring each of said patients during said baseline periodto determine a baseline quality of life; (d) dividing said plurality ofpatients randomly between a first group and a second group; (e)administering said diet program to each of said patients in said firstgroup during an intervention period; (f) maintaining each of saidpatients in said second group on a control diet with known beneficialeffects on said at least one diet-responsive condition during saidintervention period; and (g) monitoring said at least one indicator ofeach of said conditions for each of said patients after saidintervention period.

EMBODIMENTS OF THE INVENTION

In the present invention the term “semiconductor nanocrystal,” and“quantum dot” are used interchangeably herein and refer to an inorganiccrystallite between about 1 nm and about 1000 nm in diameter or anyinteger or fraction of an integer therebetween, preferably between about2 nm and about 50 nm or any integer or fraction of an integertherebetween, more preferably about 2 nm to about 20 nm (such as about2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nm). A semiconductor nanocrystal is capable of emitting electromagneticradiation upon excitation (i.e., the semiconductor nanocrystal isluminescent) and includes a “core” of one or more first semiconductormaterials, and may be surrounded by a “shell” of a second semiconductormaterial. A semiconductor nanocrystal core surrounded by a semiconductorshell is referred to as a “core/shell” semiconductor nanocrystal. Thesurrounding “shell” material will preferably have a bandgap energy thatis larger than the bandgap energy of the core material and may be chosento have an atomic spacing close to that of the “core” substrate. Thecore and/or the shell can be a semiconductor material including, but notlimited to, those of the group II-VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe,HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS,BaSe, BaTe, and the like) and III-V (GaN, GaP, GaAs, GaSb, InN, InP,InAs, InSb, and the like) and IV (Ge, Si, and the like) materials, andan alloy or a mixture thereof.

A semiconductor nanocrystal is, optionally, surrounded by a “coat” of anorganic capping agent. The organic capping agent may be any number ofmaterials, but has an affinity for the semiconductor nanocrystalsurface. In general, the capping agent can be an isolated organicmolecule, a polymer (or a monomer for a polymerization reaction), aninorganic complex, and an extended crystalline structure. The coat isused to convey solubility, e.g., the ability to disperse a coatedsemiconductor nanocrystal homogeneously into a chosen solvent,functionality, binding properties, or the like. In addition, the coatcan be used to tailor the optical properties of the semiconductornanocrystal. Methods for producing capped semiconductor nanocrystals arediscussed further below.

The term “antibody” as used herein includes antibodies obtained fromboth polyclonal and monoclonal preparations, as well as, hybrid(chimeric) antibody and, any functional fragments obtained from suchmolecules, wherein such fragments retain specific-binding properties ofthe parent antibody molecule.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. Thus, the termencompasses antibodies obtained from murine hybridomas, as well as humanmonoclonal antibodies obtained using human rather than murinehybridomas.

A semiconductor nanocrystal is “linked” or “conjugated” to, or“associated” with, a specific-binding molecule or member of a bindingpair when the semiconductor nanocrystal is chemically coupled to, orassociated with the specific-binding molecule. Thus, these terms intendthat the semiconductor nanocrystal may either be directly linked to thespecific-binding molecule or may be linked via a linker moiety, such asvia a chemical linker described below. The terms indicate items that arephysically linked by, for example, covalent chemical bonds, physicalforces such van der Waals or hydrophobic interactions, encapsulation,embedding, or the like. As an example without limiting the scope of theinvention, nanocrystals can be conjugated to molecules that can interactphysically with biological compounds such as cells, proteins, nucleicacids, subcellular organelles and other subcellular components. Forexample, nanocrystals can be associated with biotin which can bind tothe proteins, avidin and streptavidin

As used herein, a “biological sample” refers to a sample of isolatedcells, tissue or fluid, including but not limited to, for example,plasma, serum, spinal fluid, semen, lymph fluid, the external sectionsof the skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood cells, tumors, organs, and also samples of in vitrocell culture constituents (including but not limited to conditionedmedium resulting from the growth of cells in cell culture medium,putatively virally infected cells, recombinant cells, and cellcomponents).

A “small molecule” is defined as including an organic or inorganiccompound either synthesized in the laboratory or found in nature.Typically, a small molecule is characterized in that it contains severalcarbon-carbon bonds, and has a molecular weight of less than 1500grams/Mol.

In its broadest aspect, the present invention provides a compositionthat can provide information about a biological state or eventassociated with S-adenosylmethionine, S-adenosylhomocysteine andhomocysteine and C-reactive protein. The composition by way of examplecan detect the presence or amounts of the above molecules.

The composition is comprised of a fluorescent semiconductor nanocrystal(also known as a Quantum Dot) having a characteristic spectral emission,which is tunable to a desired energy by selection of the particle size,size distribution and composition of the semiconductor nanocrystal. Thecomposition further comprises a compound i.e., an antibody against SAMor SAH associated with the semiconductor nanocrystal that has anaffinity for the biological target. The composition interacts orassociates with a biological target due to the affinity of the compoundwith the target. Location and nature of the association can be detectedby monitoring the emission of the semiconductor nanocrystal.

In operation, the composition is introduced into an environmentcontaining a biological target and the composition associates with thetarget. The composition:target complex may be spectroscopically view orotherwise detected, for example, by irradiation of the complex with anexcitation light source. The semiconductor nanocrystal emits acharacteristic emission spectrum which can be observed and measured, forexample, spectroscopically.

As an advantage of the composition of the present invention, theemission spectra of a population of semiconductor nanocrystals havelinewidths as narrow as 25-30 nm, depending on the size distributionheterogeniety of the sample population, and lineshapes that aresymmetric, gaussian or nearly gaussian with an absence of a tailingregion. The combination of tunability, narrow linewidths, and symmetricemission spectra without a tailing region provides for high resolutionof multiply-sized nanocrystals, e.g., populations of monodispersesemiconductor nanocrystals having multiple distinct size distributions,within a system and enables researchers to examine simultaneously avariety of biological moieties, e.g., target analytes, tagged withnanocrystals.

In addition, the range of excitation wavelengths of the nanocrystals isbroad and can be higher in energy than the emission wavelengths of allavailable semiconductor nanocrystals. Consequently, this allows thesimultaneous excitation of all populations of semiconductor nanocrystalsin a system having distinct emission spectra with a single light source,usually in the ultraviolet or blue region of the spectrum. Semiconductornanocrystals are also more robust than conventional organic fluorescentdyes and are more resistant to photobleaching than the organic dyes. Therobustness of the nanocrystal also alleviates the problem ofcontamination of the degradation products of the organic dyes in thesystem being examined. Therefore, the present invention providesuniquely valuable tags for detection of biological molecules and theinteractions they undergo.

In one preferred embodiment, the composition comprises semiconductornanocrystals associated with molecules that can physically interact withbiological compounds. Without limiting the scope of the invention,molecules include ones that can bind to proteins, nucleic acids, cells,subcellular organelles, and other biological molecules. The compoundused in the composition of the present invention preferably has anaffinity for a biological target. In some preferred embodiments, thecompound has a specific affinity for a biological target. The affinitymay be based upon any inherent properties of the compound, such aswithout limitation, van der Waals attraction, hydrophilic attractions,ionic, covalent, electrostatic or magnetic attraction of the compound toa biological target. As used herein, “biological target” is meant anymoiety, compound, cellular or sub-cellular component which is associatedwith biological functions. The biological target includes withoutlimitation proteins, nucleic acids, cells, subcellular organelles andother biological moieties.

The ability to use semiconductor nanocrystals in order to detectmultiple targets results from their unique characteristics.Semiconductor nanocrystals have radii that are smaller than the bulkexciton Bohr radius and constitute a class of materials intermediatebetween molecular and bulk forms of matter. Quantum confinement of boththe electron and hole in all three dimensions leads to an increase inthe effective band gap of the material with decreasing crystallite size.Consequently, both the optical absorption and emission of semiconductornanocrystals shift to the blue (higher energies).

The optical properties of quantum dots are primarily dictated by theirphysical size and chemistry. Typically, electromagnetic radiation havinga wavelength within the visible light and infrared portions of thespectrum will excite quantum dots. The absorption spectrum of a quantumdot appears as a series of overlapping peaks that become increasinglylarger at decreasingly shorter wavelengths. Each peak corresponds to anenergy transition between discrete electron-hole energy states (exciton)within the quantum dot. The size of a quantum dot and the differencebetween its energy states are inversely proportional. Thus, thedifference between energy states of larger quantum dots is smaller thanthe difference between energy states of smaller quantum dots. The sizeof the quantum dots of the invention are in the range of 2-10 nm.

The smaller the difference between the energy states of a quantum dot,the “redder” (or higher wavelength) of the electromagnetic radiation(e.g., light) emitted therefrom. Thus, when excited, larger quantum dotswill emit “redder” light than smaller quantum dots, which will emit“bluer” light. As a consequence of these phenomena, the wavelength ofelectromagnetic radiation emitted by a quantum dot may be tailored byselecting the material from which the quantum dot is to be synthesizedand the size to which the quantum dot is to be synthesized. Whenexcited, known quantum dots may emit electromagnetic radiation (e.g.,light) having a wavelength from about 490 nm (blue) to about 705 nm(red).

Quantum dots have high quantum yields and resist photobleaching; theiruse therefore providing for very sensitive fluorescent biologicalassays. Different types of quantum dots are excited when exposed todifferent ranges of wavelengths of electromagnetic radiation. Currentlyavailable quantum dots may be excited by electromagnetic radiationhaving wavelengths as low as about 300 nm and as high as about 2,300 nm.

It is currently preferred that the markers within reagent solution havea Stoke's shift of about 50 nm or greater (e.g., the difference betweenexcitation of the marker at about 658 nm and emission at about 703 nm)or even of about 100 nm or greater (e.g., quantum dots that are excitedat about 405 nm may emit radiation having a wavelength of about 530 nm).

Upon exposure to a primary light source, each semiconductor nanocrystaldistribution is capable of emitting energy in narrow spectrallinewidths, as narrow as 12 nm to 60 nm, and with a symmetric, nearlyGaussian line shape, thus providing an easy way to identify a particularsemiconductor nanocrystal. It should be noted that the linewidths aredependent on the size heterogeneity, i.e., monodispersity, of thesemiconductor nanocrystals in each preparation. In addition,semiconductor nanocrystal distributions with larger linewidths in therange of 35 nm to 60 nm can be readily made and have the SAM physicalcharacteristics as semiconductor nanocrystals with narrower linewidths.

The present invention uses a composition comprising semiconductornanocrystals associated with a specific-binding molecule or affinitymolecule, such that the composition can detect the presence and/oramounts of biological and chemical compounds, detect interactions inbiological systems, detect biological processes, detect alterations inbiological processes, or detect alterations in the structure ofbiological compounds. Without limitation, semiconductor nanocrystalconjugates comprise any molecule or molecular complex, linked to asemiconductor nanocrystal, that can interact with a biological target,to detect biological processes, or reactions, as well as alterbiological molecules or processes. Preferably, the molecules ormolecular complexes or conjugates physically interact with a biologicalcompound. Preferably, the interactions are specific. The interactionscan be, but are not limited to, covalent, noncovalent, hydrophobic,hydrophilic, electrostatic, van der Waals, or magnetic. Preferably,these molecules are small molecules, proteins, or nucleic acids orcombinations thereof.

Semiconductor nanocrystal conjugates can be made using techniques knownin the art. For example, moieties generally used in the production ofsemiconductor nanocrystals, as well as other moieties, may be readilydisplaced and replaced with other functional moieties, including, butnot limited to carboxylic acids, amines, aldehydes, and styrene to namea few. One of ordinary skill in the art will realize that factorsrelevant to the success of a particular displacement reaction includethe concentration of the replacement moiety, temperature and reactivity.Thus, for the purposes of the present invention, any functional moietymay be utilized that is capable of displacing an existing functionalmoiety to provide a semiconductor nanocrystal with a modifiedfunctionality for a specific use.

The ability to utilize a general displacement reaction to modifyselectively the surface functionality of the semiconductor nanocrystalsenables functionalization for specific uses. For example, becausedetection of biological compounds is most preferably carried out inaqueous media, a preferred embodiment of the present invention utilizessemiconductor nanocrystals that are solubilized in water. In the case ofwater-soluble semiconductor nanocrystals, the outer layer includes acompound having at least one linking moiety that attaches to the surfaceof the particle and that terminates in at least one hydrophilic moiety.The linking and hydrophilic moieties are spanned by a hydrophobic regionsufficient to prevent charge transfer across the region. The hydrophobicregion also provides a “pseudo-hydrophobic” environment for thenanocrystal and thereby shields it from aqueous surroundings. Thehydrophilic moiety may be a polar or charged (positive or negative)group. The polarity or charge of the group provides the necessaryhydrophilic interactions with water to provide stable solutions orsuspensions of the semiconductor nanocrystal. Exemplary hydrophilicgroups include polar groups such as hydroxides (—OH), amines,polyethers, such as polyethylene glycol and the like, as well as chargedgroups, such as carboxylates (—CO₂ ⁻), sulfonates (SO₃ ⁻), phosphates(—PO₄ ²⁻ and —PO₃ ²⁻), nitrates, ammonium salts (—NH₄ ⁺), and the like.A water-solubilizing layer is found at the outer surface of theovercoating layer.

A displacement reaction may be employed to modify the semiconductornanocrystal to improve the solubility in a particular organic solvent.For example, if it is desired to associate the semiconductornanocrystals with a particular solvent or liquid, such as pyridine, thesurface can be specifically modified with pyridine or pyridine-likemoieties to ensure solvation.

The surface layer may also be modified by displacement to render thesemiconductor nanocrystal reactive for a particular coupling reaction.For example, displacement of certain moieties with a group containing acarboxylic acid moiety enables the reaction of the modifiedsemiconductor nanocrystals with amine containing moieties (commonlyfound on solid support units) to provide an amide linkage. Additionalmodifications can also be made such that the semiconductor nanocrystalcan be associated with almost any solid support. A solid support, forthe purposes of this invention, is defined as an insoluble material towhich compounds are attached during a synthesis sequence, screening,immunoassays, etc. The use of a solid support is particularlyadvantageous for the synthesis of libraries because the isolation ofsupport-bound reaction products can be accomplished simply by washingaway reagents from the support-bound material and therefore the reactioncan be driven to completion by the use of excess reagents.

A solid support can be any material that is an insoluble matrix and canhave a rigid or semi-rigid surface. Exemplary solid supports include butare not limited to pellets, disks, capillaries, hollow fibers, needles,pins, solid fibers, cellulose beads, pore-glass beads, silica gels,polystyrene beads optionally cross-linked with divinylbenzene, graftedco-poly beads, polyacrylamide beads, latex beads, dimethylacrylamidebeads optionally crosslinked with N—N′-bis-acryloylethylenediamine, andglass particles coated with a hydrophobic polymer.

For example, the semiconductor nanocrystals of the present invention canreadily be functionalized to create styrene or acrylate moieties, thusenabling the incorporation of the semiconductor nanocrystals intopolystyrene, polyacrylate or other polymers such as polyimide,polyacrylamide, polyethylene, polyvinyl, polydiacetylene,polyphenylene-vinylene, polypeptide, polysaccharide, polysulfone,polypyrrole, polyimidazole, polythiophene, polyether, epoxies, silicaglass, silica gel, siloxane, polyphosphate, hydrogel, agarose,cellulose, and the like.

The test strips of our invention have the configuration as shown inFIG. 1. Referring to embodiment A of figure b1, element 1 is a PVC plateincorporating a sample pad 2 for antibody conjugate layer 3. The testdevice further includes an absorption zone 7 which is typically paperand a nitrocellulose membrane 4 which includes a control band 5 and atest band 7.

In embodiment B of FIG. 1, the construction is similar to the testdevice A however it includes another test band 8 for either SAM orS-Adenosylhomocysteine.

The test strip of embodiment A of FIG. 1, includes one test band and onecontrol band. Thd test strip of embodiment B of FIG. 1 includes two testbands for SAM and SAH respectively (i.e. Methylation Index (MI) strip).The diagram of FIG. 1 shows how each component is assembled (lateralview). Liquid samples are applied through the left side of the samplepad, and the sample immediately migrates in the sample flow direction asshown in FIG. 1. The results are ready to be read in about 15 minutesafter sample application to the strips.

Numerous variations of the strip of FIG. 1 are possible. But the basicconstruction of an immunochromatographic strip is as follows and some ofthe different elements of the strip are optional and used as requireddepending on the needs of the tests.

The following describes certain elements that form part of assay devicesaccording to the present invention. Although the elements can be placedin various arrangements, according to the assay format intended and thetype of assay to be carried out, in general, the characteristics of theelements defined herein do not change between one arrangement andanother. As used herein, the elements described can be in any suitablephysical form for the purposes of assay devices according to the presentinvention, such as, but not limited to, membranes, pads, strips, orother physical forms.

A. Chromatographic Strip

As used in assay devices according to the present invention, thechromatographic strip can be composed of any suitable material that hasa high protein binding capability and supports a lateral flow assay.Typically, the chromatographic strip is a hydrophilic element and theprotein binding is through noncovalent binding. Although Applicants donot intend to be bound by this theory, current theory of binding ofproteins to nitrocellulose states that the initial interaction iselectrostatic, but subsequently hydrophobic interactions and hydrogenbonds considerably strengthen the binding. An example of achromatographic material is the commonly used nitrocellulose element,which has been treated to make it hydrophilic. Another example of achromatographic element is one made up of particles of a polymer, suchas polyethylene, fused together. The chromatographic strip is of anysize appropriate for the instrument or device used to read the resultsor for being read visually.

When antigens or antibodies are coated onto the chromatographic strip,due to its porous nature, the protein solution distributes itselfthroughout the depth of the nitrocellulose element. The proteins bind tothe pore surfaces. Because of the method of application and the physicsof the binding, more protein is bound to the top and center of the linecompared to other areas wetted by the solution used to coat the antigensor antibodies onto the chromatographic strip.

The chromatographic strip as used in assay devices according to thepresent invention includes a capture band, described further below. Thechromatographic strip also typically includes one or more control bands,also described further below.

The chromatographic strip of the present invention contains at least onecapture band for capturing the analyte and at least one control bandand, optionally, a second control band. When used in conjunction with acassette, the capture band, and the control band or bands can be viewedthrough a testing window. The capture band contains materials that arecapable of capturing an analyte in a sample if the analyte is present.For example, if the lateral flow assay is intended to measure SAM in abiological sample, the capture band will contain antibody to SAMimmobilized on the chromatographic strip at the capture band. Thechromatographic strip will additionally contain conjugates or detectableagents at the second end for detecting the captured analyte.

B. Sample Filter

Assay devices according to the present invention may employ a samplefilter (in some cases, two sample filters). The location of the samplefilter or sample filters can vary, but the sample filter is situated sothat fluid present in a sample, when applied onto the sample filter willflow from the sample filter to the chromatographic strip, eitherdirectly or indirectly. The sample filter is, in one alternative, ahydrophobic element, or alternatively a hydrophilic element or asynthetic composite of such as typically used in lateral flow assays forsample application. Examples of such sample filters include, but are notlimited to hydrophobic filters such as glass fiber filters andhydrophilic filters such as cellulose.

C. Sample Pad

In some applications, particularly when the sample does not require theremoval of cells or other large particles, a sample pad can replace thesample filter. The term “sample pad” refers to a hydrophobic element,such as a hydrophobic element, that can be used to receive a sample.

D. Conjugate Pad

The term “conjugate pad” is used to describe an element that is used inmany embodiments of assay devices according to the present invention.The conjugate pad is composed of a hydrophobic material, such as glassfiber and contains a conjugate or a detectable agent that can react withan analyte in a sample or with an analyte that is captured on thecapture band on the chromatographic strip. The detectable agentincludes, for example, antibodies or antigens specific for the analytethat are conjugated to a detectable material such as a colored material,a fluorescent material, or a chemiluminescent material or a quantum dot.An example of a colored material is colloidal gold. The conjugate padherein is of a size suitable for the chromatographic strip within theparameters described. The conjugate pads can be preblocked with a buffersolution containing trehalose and casein, although other buffersolutions can alternatively be used for preblocking. Use of theconjugate pad is not necessarily required in all embodiments of assaydevices according to the present invention. In some alternatives, theconjugate pad is omitted, and the conjugate is applied to thechromatographic strip. These alternatives are described further below.

E. Fluid Collector

The term “fluid collector” is used to describe an element used in someconfigurations of assay devices according to the present invention. Thefluid collector is typically a hydrophobic element, just like thehydrophobic element of the conjugate pad. Unlike the conjugate pad, thefluid collector does not contain any detectable agents and is used as anintermediate element, typically to transmit fluid, directly orindirectly, to the chromatographic strip.

F. Capture Band

As described above, the test strip always includes at least one captureband. The term “capture band” as used herein refers to a region or zoneon the chromatographic strip that contains at least one analyte bindingagent. The analyte binding agent is usually immobilized in a band orzone such that after reaction with a detectable agent, the band or zoneproduces an observable or measurable result reflecting the presence oramount of analyte present in the sample. The “capture band” may becomprised of more than one capture zone for capturing more than oneanalyte in the sample, in which event, more than one analyte bindingagent may be used. For example, two assay combinations that areconsidered to be within the scope of the invention as shown in theexamples.

G. Control Band

Typically, the chromatographic strip of a device according to thepresent invention also includes one or more control bands, which containcontrol agents immobilized in control binding zones.

H. Buffer Pad

Some embodiments of assay devices according to the present inventionemploy a buffer pad. The buffer pad is a hydrophilic element or asynthetic composite. The buffer pad is of a size suitable for thechromatographic strip within the parameters described.

I. Absorbent Pad or Pads

Typically, assay devices according to the present invention include oneor more absorbent pads. These absorbent pads serve to direct fluid flowwithin the device. The size and location of these absorbent pads largelydetermines the flow pattern, as described above. The absorbent pad is ahydrophilic element that can absorb liquid, such as or a cellulose-glassfiber composite. The absorbent pad herein is of a size suitable for thechromatographic strip within the parameters described.

J. Backing Pad

Some assay devices according to the present invention include a backingpad that serves as a backing for the chromatographic strip. The backingpad can be made of any inert material that is capable of supporting thechromatographic strip, such as a piece of plastic material The size ofthe backing pad is suitable for the chromatographic strip within theparameters described.

K. Fluid-Impermeable Barrier

Some embodiments of assay devices according to the present inventionincorporate a fluid-impermeable barrier interposed between elements suchas a sample filter at or near the first end of the chromatographic stripand the chromatographic strip itself.

In a preferred embodiment of the invention, immunoassays, such as ELISA(Enzyme-linked Immunosorbent Assay) for determining qualitatively andquantitatively the concentration of SAM and SAH in a biological sample,are provided in which semiconductor nanocrystal conjugates are used asthe detection reagents. The immunosorbent assay of the present inventionhas several advantages over current immunosorbent assays including, butnot limited to, simultaneous multicolor detection and, hence, multipleanalyte detection, with no requirement for enzyme development, increasedphotostability over alternative fluorophores thereby allowing increaseddetection sensitivity by virtue of the ability to monitor the signalover a long period of time, increased sensitivity over enzyme-baseddetection systems.

Semiconductor nanocrystals of varying core sizes (10-150 .ANG.),composition and/or size distribution are conjugated to specific-bindingmolecules which bind specifically to SAM and SAH. Any specificanti-analyte can be used, for example, an antibody, an immunoreactivefragment of an antibody, and the like. Preferably, the anti-analyte isan antibody. The semiconductor nanocrystal conjugates are used in animmunosorbent assay to detect any analyte for which a specific-bindingagent exists.

More specifically, the specific-binding molecule may be derived frompolyclonal or monoclonal antibody preparations, may be a human antibody,or may be a hybrid or chimeric antibody, such as a humanized antibody,an altered antibody, F(ab′).sub.2 fragments, F(ab) fragments, Fvfragments, a single-domain antibody, a dimeric or trimeric antibodyfragment construct, a minibody, or functional fragments thereof whichbind to the analyte of interest.

In this invention, we have combined together as a single unitimmunochromatographic and photochemical test strips for the simultaneousmeasurement of three critical molecules in methionine cycles, i.e. SAM,SAH and HCy, which have been reported to be very important inunderstanding the dynamics and health status of related biochemicalpathways as well as act as IVD biomarkers. The invention also deals witha new device that facilitates the aforementioned invention for thepurpose of POCT uses. The spectrometer used in the invention is acombination of a fluorescence spectrometer and an absorbance UV/VISspectrometer.

In another embodiment, the invention provides a method of determiningrisk of experiencing a major adverse cardiac event, in a patient, withinone year from presentation of at least one symptom of acute coronarysyndrome comprising the steps of: (a) obtaining a test sample from saidpatient; (b) determining the amount of at SAM, SAH, HCy and C reactiveprotein using a quantum dot based assay employing animmunochromatographic strip; (c) calculating the MI in said test sample;and c) comparing the amount of said four biomarkers to biomarkerreference standards, wherein said risk is determined by results of saidcomparison.

By way of example, the preparation and assemblage of the immunoassaytest immunochromatographic strip is done as follows. Briefly, goatanti-mouse IgG and BSA-SAH were separately applied to NCM (2.5×2.0 cm)with 3.5 μg in 10 mM phosphate-buffered saline, pH 7.4, to be used asthe control zone and the test zone. The distance between the controlzone and the test zone was 0.5 cm. The NCM was then dried for 1.5 hoursat 37° C. to fix the antibody and antigen. The NCM was pasted onto thepolyvinyl chloride strip with the adsorption pad on the top end, and thequantum dot-conjugated pad overlapped by the sample pad was adhered tothe bottom end of the NCM. The quantum dot-conjugated pad had beenprepared by adding the anti-SAH MoAb-coated quantum dots (i.e, CdSeNPs)to the glass fiber (2.5×1.0 cm). The resultant conjugated pad wasincubated at 37° C. for 1.5 hours until fully dried. The sample pad ofglass fiber (2.5×2.0 cm) was submerged in 10 mM phosphate-bufferedsaline, pH 7.4 and containing 0.05% Tween 20, and dried at 37° C. for1.5 hours. Finally, the test device was cut into 5 mm-wide strips andstored at RT before use.

When using lanthanide based fluorescent molecules, the SAM or SAHbinding antibody is conjugated with a fluorescent label such as, withoutlimitation, the rare earth chelates (e.g., europium chelates). Thefluorescent labels can be conjugated to the antibody using conventionaltechniques in immunology. Fluorescence can be quantified using afluorimeter or UV/vis spectrophotometer using the known extinctioncoefficient of the fluorescent label.

The fluorescence properties of certain lanthanide chelates, especiallychelates of europium and terbium, are well suited fluorescent markers.The absorbance of these chelates is very strong, (more than 10⁴) anddependent upon the ligands. Although the quantum yield is often smallerthan that for organic markers these chelates have other advantages, thusthe emission appears at relatively long wavelengths (terbium 544 nm,europium 613 nm) in which wavelength range the serum fluorescence is lowand furthermore the excitation maximum is within the short UV-range(Terbium-chelates 270-320 nm, Eu-chelates 320-360 nm) independent of theligands which makes it possible to excite them with lamps or laserscommercially available and furthermore the Stoke's shift is very long(240-270 nm) and the emission band is sharply limited which enables asmall band width. The most essential property is however that thefluorescence time is long, about 50-1000 microseconds which makes itpossible to use the above mentioned instrumentation. As the fluorescenceis measured with a certain delay during which the backgroundfluorescence has decayed, the effect of an unspecific backgroundradiation can be eliminated.

The chelates of europium and to a certain extent terbium together withdifferent .beta.-diketones are the most used chelates due to theirability to laser in different solutions and at different temperatures.The most widely used β-diketones are benzoylacetone (BA),dibenzoylmethane (DBM), thenoyltrifluoroacetone (TTA),benzoyltrifluoroacetone (BTA), 1- and 2-naphthoyltrifluoroacetone(1-/2-NTA), acetylacetone (AcA), trifluoroacetylacetone (TFAcA), andhexafluoroacetylacetone (HFAcA).

The strong fluorescence of the lanthanide chelates is due to theabsorption by the ligands of the excitation radiation and of the energytransfer from the triplet state of the ligand which gives rise to anarrow band radiation with a long wavelength characteristic for metals.

Before a chelate of the above mentioned type could be used as afluorescent marker it has to be attached to the antibody/antigen to beinvestigated. Furthermore, the metal has to give a fluorescent radiationalso after the binding and in a water solution. To be stable enough,also in very diluted form (even below 10⁹ M) and under conditions whereother chelate forming reagents are present as well as an excess of othermetal ions, the binding system must be very strong. The stabilityconstant of the chelate must be well above 10¹⁰ and additionally thebinding ligand has to leave coordination positions free for anotherbidentate ligand.

By way of further background into the present invention, given theimportant roles of SAM, SAH, HCy and C-reactive proteins in variouspathological processes, it is desirable to conveniently measure thelevels of SAM, SAH, HCy and C-reactive protein using the methods thatcan be done in common research and clinical labs. With the availabilityof specific antibodies against SAM, SAH and C-reactive protein variousforms of immunoassays using immunochromatographic test strips areextremely useful in the clinical environment. Having a test strip thatmeasures SAM, SAH, HCy and C-reactive protein would be an ideal additionto the clinical lab.

In a further embodiment of the invention, with the fluorescence-labeledanti-SAM and anti-SAH antibodies of the invention that have been provento be specific, quick and easy measurements of SAM and SAH can beperformed at the cellular level via flow cytometry, immunofluorescencemicroscopy or LSCM. The immunofluorescence microscopy has the advantageof studying the levels and locations of SAM and SAH even with a smallnumber of cells, e.g. studying SAM and SAH from cells in their earlystages of embryo development with a couple of hundreds of cells or evenless. The LSCM results from FIG. 7 showed that intracellularlocalizations of SAM and SAH were somewhat similar. SAM and SAH wereseen mostly in mitochondria, peri-nuclei and in nucleoli. In HepG2 cellscultured for 40 h, compared to L02, obviously reduced levels of SAM andSAH were observed in cytoplasm, slightly more SAH and SAM in nuclei(consistent with FCM results from FIG. 5) yet they were not focused innucleoli area as L02 cells were.

The invention also provides an easy and quick homogeneous immunoassaythat does not have special strip preparation as well as no washing andseparation steps that can also be used conveniently in the point-of-caretest (POCT) setting besides the commonly known dry test strips. FIG. 8show simple diagrams illustrating how the two formats of TR-FRETtechnology may be used in the quantitative measurement of SAM and SAHusing the bio-conjugates described in this invention. With format A ofFIG. 8, specific antibodies against SAM or SAH are associated withacceptor dyes directly or indirectly through rabbit or goat anti-mouseIgG that is labeled with acceptor dye. Two tracing methods, SA-biotinand Dig-anti-digoxin antibody specific binding partners, are shown thatare conjugated to donor dyes. The biotin-conjugated (or Dig-conjugated)SAM or SAH with different linkers brings donor and acceptor dyestogether in close proximity, most likely less than 100 angstrom (A),which allows the donors to excite the acceptor dyes. The energy transferwith the donors occurs and a distinguished fluorescence emitted at aspecific wave length from acceptor dyes is measured that reflects onlythe portion of the molecules that are able to connect donors andacceptors together specifically. Free SAM or SAH molecules from a samplecompete with the bio-conjugates for binding the anti-SAM or anti-SAHantibodies, therefore lead to reduced fluorescent signals. Competitivemeasurement can be established based on the competitive bindingcharacteristics.

With format B of FIG. 8 SAM, SAM analog or SAH is conjugated (with orwithout a linker) to an acceptor dye, which will compete with free SAMor SAH from samples for binding to the antibodies against SAM or SAHthat are attached to donor indirectly through rabbit or goat anti-mouseIgG. The emitted fluorescence from acceptor dyes reflects the amounts ofSAM or SAH bound to the donor dyes that are not competed by the SAM orSAH in the samples, i.e. donor-specific antibody-antigen-acceptorcomplex. The amount of specific antibodies that bind to un-conjugatedSAM or SAH molecules will not have fluorescence to be read, whichconstitutes one of the competing parties in the competitive assay. Freeanti-SAM or SAH antibody, if any, which is not conjugated with donordyes, will consume either labeled or unlabeled antigens. Both donor andacceptor fluorescence signals are read with the TR-FRET microplatereader and the acceptor fluorescence/donor fluorescence can becalculated that will be used in quantifying SAM or SAH from a sample.

BRET (Bioluminescence Resonance Energy Transfer) technology is similarto TR-FRET or FRET except for the donor dye is replaced withbioluminescent enzyme, e.g. luciferase (EC1.13.12.7) or Luc. Theacceptor dye should be chosen so that it has an optimal spectral overlapbetween the Luc bioluminescent spectra and the dye excitation spectraand higher quantum yield. For example, SAM or SAH (antigen) isconjugated to Luc, the fluorescent dye that meets the criteria above isconjugated to the anti-SAM or anti-SAH antibody. Addition of fireflyluciferin, a Luc substrate, causes luciferin to luminescence andmeanwhile excites acceptor dyes to emit fluorescence whenLuc-antigen-antibody-acceptor dye complex is formed. Both donorluminescence and acceptor fluorescence are recorded and BRET index(acceptor fluorescence/donor luminescence) can be calculated. The morethe SAM or SAH antigens from a sample are present, the less the acceptorfluorescence, thereby the less the BRET index.

Competitive BRET homogeneous immunoassay can be established to quantifySAM or SAH after optimizing every condition so the linearity,sensitivity, recoverability and reproducibility are satisfactory. A partof the FIG. 8A also illustrates how this process works. The BRET-basedmethod does not require laser excitation of donor dye at the time ofdetection. Instead it only needs to add the substrate of the luciferase.When enough substrates start to generate luminescence that can bemeasured, it also excites the acceptor fluorescent materials that arebrought to its close proximity by specific antigen-antibody. It does notexcite acceptor fluorescent dyes that are not associated with luciferasedonor. Therefore, the emission signals measured reflect the part ofantigen-antibody complex containing both the donors (bio-conjugates) andacceptors, not the SAM or SAH antigens from samples or standards thatare only associated with acceptors via antibodies.

EXAMPLES

The following examples are intended to demonstrate the usefulness of themethods and compositions of the present invention and should not beconstrued to limit the scope of the invention in anyway. In the presentspecification the term biological sample is intended to include saliva,urine, blood, serum, plasma, brain fluids, cerebrospinal fluids, tissuesamples and cells or anything derived from the body of a mammalincluding a human.

The quantum dots (CdTe/CdSe, CdHgTe/ZnS, etc.) with mean diameter of2-10 nm were purchased from NN-Labs, LLC (Fayetteville, Ark. 72701).Fluorescent dye Europium chelates or other lanthanide metals, etc. withmean diameter at 200 nm-300 nm were purchased from Bangslab (Fishers,Ind. 46038). In the context of the present specification we refer to thelanthanides fluorescent dyes and quantum dots to as fluorescent tracers(FTs). Except for the methods of conjugation of different FTs toantibodies, other procedures including standard curves for making testsstrips are the SAMe between quantum dots and lanthanide chelates.Conjugation of quantum dots to antibodies was carried out using QuantumDot Labeling Kit (Cat #Q0101, NajingTech, Hangzhou, China).

Example 1—SAM Quantitative Tests Format 1: A Homogeneous Immunoassay fora Quick Quantification of SAM

Employ the homogeneous immunoassay such as Homogeneous Time-ResolvedFluorescence (HTRF® technology, as exemplified in our application Ser.No. 15/091,544 filed Apr. 5, 2016, the entire contents of which areincorporated by reference herein as if they were entirely denoted) andthe competitive method to quantify SAM from samples by using anti-SAMmonoclonal antibody and bio-conjugates (as exemplified in ourapplication Ser. No. 15/091,544 filed Apr. 5, 2016, the entire contentsof which are incorporated by reference herein as if they were entirelydenoted.)

(1) Use of the biotin, digoxigenin or digoxin conjugated SAM or SAManalogs as well as d2-conjugated SAM or SAM analogs with differentlengths of linkers in the methods described in FIG. 8 on HTRF®

Rabbit anti-mouse IgG-XL665 and Europium (Eu3+) cryptate labeling kitwere purchased from Cisbio Bioassays. Label mouse anti-digoxin oranti-digoxigenin antibody (anti-Dig antibody, PerkinElmer) to Eu3+cryptate. Optimize the dosage of each of the following components:Digoxin(Digoxigenin)-6C-aza-SAM, anti-Digoxin(Digoxigenin)-antibody-Eu3+cryptate, mouse-anti-SAM antibody clone 118-6 (a sample of the subjecthybridoma clone 118-6 was deposited at the China Center For Type CultureCollection (CCTCC) Address: Wuhan University, Wuhan Zip code: 430072 onSep. 16, 2017, and has been assigned the CCTCC number C2017179) andrabbit anti-mouse IgG-XL665 in a buffer containing 100 mM PB, pH 7.0,0.1% protease-free BSA, 100 mM KF, 0.1% Tween 20. In a competitive HTRFassay, SAM standard is used in the range of 0-3000 nM. The test isperformed with a micro-titer strip of 1-10 wells to a final volume of100 μl/well. All assay components are combined and incubated for about30 min at room temperature. The assay plates are read with a smallpoint-of-care micro-titer strip reader for HTRF assays. Time-resolvedfluorescence is measured at a 50 μs delay after each excitation pulse.Emissions are measured at 665 nm for detection of the FRET signal (Acounts), and at 620 nm for detection of the Eu(K) signal (B counts). TheB counts should be the same for all assay wells, which act as aninternal control and indicator of the absorbance of the background. Thefluorescent signals are measured simultaneously, and the ratio ((Acounts−10,000)/B counts) is reported. This ratio is minimally affectedby absorbance as both the 665 nm and the 620 nm signals are impactedsimilarly. The ratio and the concentration of the SAM standards are usedto plot the standard curve. The more the SAM is from a sample, the lowerthe A counts and hence the ratio.

(2) Use of the Luciferase-6C-aza-SAM in BRET

Mouse anti-SAM antibody from clone 118-6 (A sample of the subjecthybridoma clone 118-6 was deposited at the China Center For Type CultureCollection (CCTCC) Address: Wuhan University, Wuhan Zip code: 430072 onSep. 16, 2017, and has been assigned the CCTCC number C2017179) wasconjugated to Alexa Fluor 610-x using fluorescent antibody labeling kit(Thermo-Fisher). Optimize the molar ratio of the bio-conjugate toluciferase, molar ratio of mouse anti-SAM antibody to Alexa Fluor 610-x,the working concentrations of Luciferase-6C-aza-SAM (donor Luc-SAM),mouse anti-SAM antibody from clone 118-6 (A Culture Collection (CCTCC)Address: Wuhan University, Wuhan Zip code: 430072 on Sep. 16, 2017, andhas been assigned the CCTCC number 02017179) (acceptor FL-Ab) and thecompeting SAM from a sample or standard in a buffer containing 100 mMPB, pH 7.0, 0.1% protease-free BSA, 100 mM KF, 0.1% Tween 20. In acompetitive BRET assay, SAM standard is tested in the range of 0-3000nM. The test is performed with a micro-titer strip of 1-10 wells to afinal volume of 100 μl/well. Three assay components above and thesubstrate luciferase are combined and incubated for 15-30 min at roomtemperature. The assay plates are read with a small point-of-caremicro-titer strip reader for BRET assays. Time-resolved fluorescence ismeasured at a 50 μs delay after each excitation pulse. Emissions aremeasured at 630 nm for detection of the BRET signal, and at 550 nm fordetection of the luciferin signal. Find the proper molar ratio of TheBRET index (FL-Ab/Luc-SAM). With the right Luc-SAM (molar ratio Luc:SAMas 1:20) and FL-Ab (molar ratio FL:Ab as 4-8:1) conjugates, the amountof antibody bound is in linear relationship with BRET index, the BRETindex and the concentration of the SAM standards are used to plot thestandard curve. The more the SAM is from a sample, the lower the BRETindex.

Format 2: A Fluorescent Immunochromatographic Strip for a QuickQuantification of SAM

(1) Conjugation of monoclonal antibody against SAM to fluorescenttracers and then applied the conjugate evenly to 33GLASS (GE HealthcareBiosciences Corp. Piscataway, N.J.): The uniform europium dyedmicrospheres (0.20 μm diameter polymer P(S/V—COOH), Bangs Laboratories.Inc. Fishers, Ind.) were washed twice with MES (2-N-morpholinoethanesulfonic acid) at 14,000 rpm centrifugation for 10 minutes. AddedEDC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide) to 1.5 mg/ml,N-hydroxysuccinimide (NHS) to 2 mg/ml to activate the polymer. Addedanti-SAM antibody from clone 84-3 (A sample of the subject hybridomadone 84-3 was deposited at the China Center For Type Culture Collection(CCTCC) Address: Wuhan University, Wuhan Zip code: 430072 on Sep. 16,2017, and has been assigned the CCTCC number 2017178), (Cat# MA00202,Arthus Biosystems, VA) at the final concentration of 40 μg/ml and shakenat room temperature for 2.5 h. The conjugate was stored in 20 mM Trisbuffer with 0.5% BSA and EDTA-Na2, applied evenly to the glass fiberafter proper dilution at the density of 4 ul/cm, followed by drying at37° C. for 12 h.

(2) Immobilized BSA-SAM at 0.2 mg/ml for test line (T) and goatanti-mouse antibody for control line (C) at 1.2 mg/ml onto anitrocellulose membrane: The reagents were immobilized with 50 mMphosphate buffer, pH 7.4. The membranes were dried at 56° C. overnightand then assembled with the sample pad and the adsorption membrane. Theresulting multi-membrane composite was cut into 3.8-mm test strips. Thetest strips were packed in a specialized black PVC cassette and thenplaced to a sealed aluminum foil bag containing silica gel as adesiccant.

(3) Sample pad was processed with anti-RBC (red Blood Cell) antibody,Tween 20, BSA and EDTA-Na2 in 50 mM Tris buffer so that all blood sampletypes can be used. The composition of a test strip is illustrated inFIG. 1A.

(4) Measurement: About 100 μl of plasma or serum sample or 50 μl wholeblood plus 50 μl dilution buffer was added to the sample well of thetest strip cassette. In 15 minutes, insert the cassette into the slot ofa fluorescence reader (with 365 nm excitation light). The fluorescenceintensity was measured, which would be converted into actual levels ofSAM based on the preinstalled standard curve (FIG. 2) calculated andupdated per batch of strips. For this particular strip, the standardcurve is shown in FIG. 2, where the x-axis is base 10 logarithm of theconcentration of SAM ranging from 0 to 3000 nM. The y-axis is the base10 logarithm of the ratio of fluorescent signal of test line (T) to thatof control line (C).

Example 2—SAH Quantitative Tests Format 1: A Homogeneous Immunoassay fora Quick Quantification of SAH

Employ the homogeneous immunoassay such as Homogeneous Time-ResolvedFluorescence (HTRF® technology, as exemplified in our application Ser.No. 15/091,544 filed Apr. 5, 2016, the entire contents of which areincorporated by reference herein as if they were entirely denoted) andthe competitive method to quantify SAH from samples by using anti-SAHmonoclonal antibody and bio-conjugates (as exemplified in ourapplication Ser. No. 15/091,544 filed Apr. 5, 2016, the entire contentsof which are incorporated by reference herein as if they were entirelydenoted.). The uses of the biotin, digoxigenin or digoxin conjugatedSAH, d2-conjugated SAH with different lengths of linkers in HTRF®, andluciferase conjugated SAH in BRET with different lengths of linkers inthe methods described in the FIG. 8 are similar to the proceduresdescribe in the Example 1 Format 1 in this invention except for usinganti-SAH antibody and SAH when anti-SAM antibody and SAM (or SAManalogs) were used.

Format 2: A Fluorescent Immunochromatographic Strip for a QuickQuantification of SAH

Used the same procedure as Example 1 above, a mouse anti-SAH antibody301-3 (Cat #MA00303, Arthus Biosystems, VA) was used at the finalconcentration of 80 μg/ml. The standard curve for this particular stripis shown in FIG. 3, where x-axis is base 10 logarithm of theconcentration of SAH ranging from 0 to 3000 nM. The y-axis is the base10 logarithm of the ratio of fluorescent signal of test line (T) to thatof control line (C).

Example 3—MI Strip A Fluorescent Immunochromatographic Test Strip forMeasuring Methylation Index (MI)

Using the method of Example 1 as described above but BSA-SAM (or SAManalog) and BSA-SAH were applied to different areas of the NC membraneand dried. Both FT-anti-SAM and FT-anti-SAH were absorbed evenly to theglass fiber, and then assembled as shown in the FIG. 1B. Thefluorescence intensity of the FT was measured separately, which will beconverted into actual levels of SAM and SAH based on the preinstalledstandard curves for the batch of strips. SAM and SAH test lines willdisplay as two SAMe or different colors depending the type of FTs usedto label the anti-SAM and anti-SAH antibodies. The strip allowsmeasuring SAM and SAH at the SAMe time quickly and easily. MI iscalculated and displayed on the Dry Immunofluorescence Analyzer.

Example 4—CRP Quantitative Strip A Fluorescent ImmunochromatographicStrip for a Quick Quantification of Full CRP

(1) Conjugation of monoclonal antibody against SAM to fluorescenttracers and then applied the conjugate evenly to 33GLASS (GE HealthcareBiosciences Corp. Piscataway, N.J.): The uniform europium dyedmicrospheres (0.20 μm diameter polymer P(S/V-COOH), Bangs Laboratories.Inc. Fishers, Ind.) were washed twice with MES (2-N-morpholinoethanesulfonic acid) and separated at 14,000 rpm centrifugation for 10minutes. Added EDC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide) tofinal concentration of 1.5 mg/ml, N-hydroxysuccinimide (NETS) to 2 mg/mlto activate the polymer. After washing with MES, microspheres wasreconstituted in 500 μl MES pH 6.0. Added anti-CRP antibody M-5191(Biobridge, Beijing, China) 7 μl (2.82 mg/ml) and shaken at roomtemperature for 2.5 h. The conjugate was stored in 20 mM Tris bufferwith 0.5% BSA and EDTA-Na₂, applied evenly to the glass fiber after 1:3dilution at the density of 4 ul/cm, followed by drying at 37° C. for 18h.

(2) Immobilized anti-CRP antibody M-5192 (Biobridge, Beijing, China) at0.05 mg/ml for the first test line (T1) and 0.4 mg/ml for the secondtest line (T2), goat anti-mouse antibody for control line (C) at 1.2mg/ml onto a nitrocellulose membrane: The reagents were immobilized with50 mM phosphate buffer, pH 7.4. The membranes were dried at 56° C.overnight and then assembled with the sample pad and the adsorptionpaper. The resulting multi-membrane composite was cut into 3.8-mm teststrips. The test strips were packed in a specialized black PVC cassetteand then placed to a sealed aluminum foil bag containing silica gel as adesiccant.

(3) The composition of the test strip is illustrated in FIG. 1B withouta sample pad as blood samples will be diluted at about 600 folds beforetesting.

(4) Measurement: About 100 μl of diluted plasma or serum sample or wholeblood plus was added to the sample well of the test strip cassette. In15 minutes, insert the cassette into the slot of a fluorescence reader(with 365 nm excitation light). The fluorescence intensity was measured,which would be converted into actual levels of CRP based on thepreinstalled standard curve (FIG. 4) calculated and updated per batch ofstrips.

The standard curve for this particular strip is shown in FIG. 4, wherethe x-axis is the concentration of CRP ranging from 0 to 130 mg/L. They-axis is the ratio of fluorescent signal of the second test line (T2)to that of control line (C).

Example 5—HCy Quantitative Tests Format 1: A Homogeneous Immunoassay fora Quick Quantification of HCy

Employ the homogeneous immunoassay such as Homogeneous Time-ResolvedFluorescence (HTRF® technology, as exemplified in our application Ser.No. 15/091,544 filed Apr. 5, 2016, the entire contents of which areincorporated by reference herein as if they were entirely denoted) andthe competitive method to quantify HCy from samples either by usinganti-HCy monoclonal antibody or by measuring the level of SAH that isgenerated from the biochemical reaction describe in the Example 6. Themethod to measure SAH is the same as the procedure descried in theExample 2 Format 1.

The uses of the biotin, digoxigenin or digoxin conjugated HCy,d2-conjugated HCy with different lengths of linkers in HTRF®, andluciferase conjugated HCy in BRET with different lengths of linkers inthe methods described in the FIG. 8 are similar to the proceduresdescribe in the Example 1 Format 1 in this invention except for usinganti-HCy antibody and HCy when anti-SAM antibody and SAM (or SAManalogs) were used.

Format 2: A Fluorescent Immunochromatographic Strip for a QuickQuantification of HCy

Employ the similar method as in the Example 1 Format 2 to quantify HCyfrom samples either by using anti-HCy monoclonal antibody or bymeasuring the level of SAH that is generated from the biochemicalreaction describe in the Example 6.

Example 6—HCy Qualitative Strip An Immunochromatographic Test Strip fora Quick Qualitative Measurement of HCy

Materials:

Reagent Stock Solution Reagent Stock Solution DTT 100 mM Boric acid 0.2MpH 8.2 buffer SAH-Na 100 uM Tris buffer 0.1M pH 8.2 HMT 0.2 mg/ml in 30%PBS 0.1M pH 7.4 glycerol SAM 100 uM K₂CO₃ 0.2M Homocysteine 100 uMD-Trehalose 30% Goat-anti-mouse IgG 7.8 mg/ml sucrose 50% Anti-SAHantibody 5.1 mg/ml BSA 10% SAH-BSA 3.73 mg/ml colloidal 70 nm in golddiameterOther materials include glass fiber K88 (Tongcheng Paper Production, Co,Ltd, Anhui, China), nitrocellulose membrane, Trion X-100, Tween 20,casein, fetal bovine serum and PVP (Polyvinylpyrrolidone). Mouseanti-SAH antibody (Cat #MA00307, Arthus Biosystems, VA).

HCy plasma or serum samples underwent some chemical reactions so thatall HCys were freed from protein associations and in a reductive formbefore they were converted to SAH as follows:

Homocysteine+S-adenosylmethionine—(HMT)→S-adenosylhomocysteine+Methionine,whereas HMT is homocysteine methyltransferase. Test reaction: 3 μl HCy,3 μl SAM, 3 μl HMT and 91 ul 100 mM PBS, pH7.4; control reaction: 3 μlHCy, 3 μl SAM, 30% glycerol and 91 ul 100 mM PBS, pH7.4. Thoroughlymixed and let it react for 5 min and then added 80 μl to SAH test strip.

The reaction product SAH is measured with a qualitative SAH strip with aproper cutoff value that reflects the cutoff value of limiting materialHCy in human plasma or serum, i.e. normal subjects have HCy at 10 μM andbelow; patients with abnormal HCy that is higher than 15 μM. Therefore,we made a colloidal gold SAH test strip that shows test line (T) andcontrol line (C) with the following readout:

C line does not have any colloidal gold signal: the strip is invalid.

Both T and C have the similar colloidal gold signal intensity: HCy levelfrom a sample <10 μM;

The C has much stronger colloidal gold signal than T line and T line isbarely visible: HCy level from a sample >=15 μM;

The C has stronger colloidal gold signal than T line and T line isvisible: HCy level from a sample is between 10 to 15 μM;

The SAH test strip was made according to the following procedure:

-   -   (1) 1 ml 70 nm colloidal gold, 27 μl K₂CO₃, 8 μl mouse anti-SAH        antibody, rested for 20 min, then added 100 μl 10% BSA, rested        for 15 min followed by centrifuging at 12,000 rpm for 15 min.        Discarded supernatant. Washed with 20 mM boric buffer containing        1% BSA, D-Trehalose and sucrose once, and reconstituted in 120        μl boric buffer.    -   (2) Applied the conjugated antibody at 4 μl/cm to glass fiber        K88 evenly.    -   (3) Sample pad was processed with 100 mM Tris pH 8.2 containing        0.1-2% PVP, Triton x-100 and casein. Dried at 37° C. overnight.    -   (4) Test strip was assembled on a PVC plate according to the        method illustrated in the FIG. 1A.

Example 7—MIHC Strip An Immunochromatographic Test Strip forSimultaneous Measurement of SAM, SAH and HCy

MIHC1 represents a methylation index and homocysteine triple test stripformat 1. The unit consists of two test strips. (a) One is an MI stripas in Example 3. (b) The other one is an HCy strip as in Example 5.

MIHC2 represents a methylation index and homocysteine triple test stripformat 2. The unit consists of two test strips. (a) One is an MI stripas in Example 3. (b) The other one is an HCy strip as in Example 6.

The accompanying device is able to read, process and output the resultsat the SAM time reporting the values of SAM, SAH, MI and HCy from asample qualitatively or/and quantitatively.

Example 8—MIHCR Strip An Immunochromatographic Strip for SimultaneousMeasurement of SAM, SAH, Homocysteine (HCy) and CRP

MIHCR1 represents a methylation index, homocysteine and C-reactiveprotein quadruple test strip format 1. The unit consists of three teststrips. (a) One is an MI strip as in Example 3. (b) The second one is anHCy strip as in Example 5. (c) The third one is the CRP strip as inExample 4.

MIHCR2 represents a methylation index, homocysteine and C-reactiveprotein quadruple test strip format 2. The unit consists of three teststrips. (a) One is an MI strip as in the Example 3. (b) The second oneis an HCy strip as in Example 6. (c) The third one is the CRP strip asin Example 4.

The accompanying device is able to read, process and output the resultsat the SAM time reporting the values of SAM, SAH, MI, CRP and HCy from asample qualitatively or/and quantitatively.

Example 9—SAM Semi-Quantitative Strip Colloidal Gold or ColloidalMicrosphere Semi-Quantitative Test Strip for SAM

The test unit does not require any device to read results. Triple teststrips are assembled into one single unit with each strip havingdetection band at cutoff values of 50 nM, 400 nM, 800 nM respectively.The cutoff values can be changed to different values besides 50 nM, 400nM and 800 nM. The value from a blood sample can be read out as thefollowing: <50 nM; 50-400 nM; 400-800 nM; >800 nM. Colloidal gold ormicrospheres were used to label mouse anti-SAM antibody (Cat #MA00201,Arthus Biosystems, VA). Conjugation of antibody was similar to that inExample 6. Assembling of the test strip is the SAM as in shown FIG. 1Aand in the Example 1. Colloidal gold or microsphere results can be seenwith naked eyes. Therefore, this method is quick, easy andcost-effective without having to use any additional device.

Example 10—SAH Semi-Quantitative Strip Colloidal Gold or ColloidalMicrosphere Semi-Quantitative Test Strip for SAH

The test unit does not require any device to read results. Triple teststrips are assembled into one single unit with each strip having adetection band with cutoff values of 200 nM, 600 nM, 1200 nMrespectively. The cutoff values can be changed to different valuesbesides 50 nM, 600 nM and 1200 nM. The value from a blood sample can beread out as the following: <200 nM; 200-600 nM; 600-1200 nM; >1200 nM.Colloidal gold or microspheres were used to label mouse anti-SAHantibody 839-6 (Cat #MA00307, Arthus Biosystems, VA). Conjugation ofantibody was similar to that in Example 6. Assembling of the test stripis the SAM as in shown FIG. 1A and in the Example 1. Colloidal gold ormicrosphere results can be seen with naked eyes. Therefore, this methodis quick, easy and cost-effective without having to use any additionaldevice.

Example 11—Other Immunoassay Systems

Besides using dry test strip format as described above, FTs can also beused in other aqueous systems as tracers in a list of potentialmeasurements below.

FT-anti-SAM and FT-anti-SAH antibodies can be used in cell-basetechnologies such as flow cytometry and immunofluorescence microscopy toinvestigate the metabolism, dynamics, distribution and levels of SAM andSAH within cells, tissues and organs under different scenarios.

A. FCM (Flow Cytometry)

MAT activity was stimulated by Met in cells using FCM. SAM and SAH weredouble stained and analyzed from cells after cultured and treated asindicated in FIG. 5. The FCM results are consistent with the resultsform ELISA (data not shown) and LSCM yet FCM provides more informationabout changes of SAM levels in nucleus and cytoplasm compartments. Theeffects of Met-stimulated MAT activities have similar pattern forcytoplasm and nucleus, which is different from the effects on primaryliver cells. Higher dosage of Met (1 mM) inhibits instead of stimulates(as in 0.5 mM Met) MAT activity in L02 cells in nuclei whereas 1 mM Metcontinuously stimulates MAT activity in cytoplasm of L02 cells. Metinhibits MAT activity in HepG2 both in cytoplasm and nucleus and thusSAM is decreased (FIG. 5A). In both cell lines, nuclear SAM constitutes80-85% of the total SAM and methylation indices are similar too. Innormal mouse liver cells, about 4.6% of SAM is located in nucleus. With1 mM Met-stimulation for 24 h, nuclear SAM level is increased by 4folds, constitutes about 22.5% of the total SAM. Met-stimulated MATcauses nuclear SAM to increase whereas cytoplasm SAM is decreased within1 mM Met dosage. Primary liver cells were cultured in Met-free mediumfor 20 h, MAT activity was induced and SAM was increased in nucleus butwas reduced in cytoplasm (FIG. 6). This indicated critical roles thatSAM needs to play are in nucleus in response to Met hunger/deficiency(regulated expressions of certain genes). In the current testconditions, cytoplasm and total cell methylation index (MI) was1.85-2.55, but the MI was 0.3 in normal liver nucleus that was increasedto 1.2 after stimulation with 1 mM Met. Nuclear MI was 0.98 in Met-freemedium cultured for 20 h, which is about 3-fold higher than normal livercells and is consistent with the changes of SAM levels.

Two different types of fixation/permeabilization buffers were tested forall cell types, i.e. nuclear fixation/permeabilization buffer (Cat#00-5523 FoxP3_TF Staining Buffer Set, eBioscience, San Diego, Calif.)by which both cytoplasm and nucleus targets were stained andintracellular fixation/permeabilization buffer was used (Cat #00-8824,eBioscience, San Diego, Calif.) by which only cytoplasm targets weremeasured.

(1) Prepare cell suspension according to the protocol of cell digestionwith trypsin;

(2) Centrifuge cell suspension at 1500 rpm for 5 minutes and abandon thesupernatant;

(3) Re-suspend the cells with at least 1 ml PBS in about 106cells/sample;

(4) Centrifuge at 1500 rpm for 5 minutes and abandon the supernatant;

(5) Add 100 μl fixation buffer to each sample (if the fixation bufferwas 4% paraformaldehyde, add 400 μl/sample). Keep the samples in dark atroom temperature for 30 minutes, and then centrifuge the suspension at1500 rpm for 5 minutes and abandon the supernatant;

(6) Wash with 100 μl permeabilization buffer and then centrifuge thesuspension and abandon the supernatant;

(7) Incubate with 100 μl permeabilization buffer at room temperature for20 minutes, and then centrifuge the suspension and abandon thesupernatant;

(8) Re-suspend with 100 μl permeabilization buffer. Add 10 μlfluorescence labeled antibodies and incubate it for 30 minutes.

(9) Wash with PBS for twice and re-suspend the cells in 0.5 ml PBS formeasurements with BD FACSCanto II Flow Cytometer. The results are shownin FIGS. 5 and 6.

FIG. 5 shows the flow cytometry (FCM) results from cells double stainedwith Alexa Fluor 647 conjugated anti-SAM 118-6 antibody (Cat #MAF00201,Arthus Biosystems, VA) at 4.5 μg/ml while FIG. 6 shows FCM results fromcells double stained with Alexa Fluor 488 conjugated anti-SAH antibody301-3 (Cat #MAF00301, Arthus Biosystems, VA) at 45 μg/ml. Both SAM andSAH levels from cytoplasm and nucleus compartments are shown. Normalliver cell line L02 and hepatocellular carcinoma cell line HepG2 weretreated with 0, 0.5 mM and 1 mM methionine (Met) for 24 h. Mouse primaryliver cells were isolated and treated with 0, 0.5 mM, 1 mM Met for 24 hand cultured in Met-free MEM medium for 20 h. FIG. 5 shows SAM levelswhile FIG. 6 shows SAH levels.

B. Immunofluorescence Laser Scanning Confocal Microscopy (LSCM)

(1) Cleaned with alcohol the special pieces of glasses for LSCM (thatare designed to allow cells to grow on easily and be taken photos undermicroscope). Placed under UV light in the hood for at least 10 minutes.

(2) Put the glasses into 24-well cell culture plate using steriletweezers. Seed proper amount of cells (normal liver cell line L02 andhepatocellular carcinoma cell line HepG2) based on the knowledge of cellgrowth rate (e.g. 5×104/well). Culture for 24 h with or without anytests designed, e.g. methionine stimulation.

(3) When cells were ready to be stained, removed medium from wells andwashed with 1 ml 1×PBS for 3 times.

(4) Added 200 μl 80%-20° C. stored acetone to fix the cells under −20°C. for 30 minutes.

(5) Wash with 1 ml 1×PBS for 3 times.

(6) Added Alexa Fluor-488-anti-SAH antibody at 40 μg/ml and Alexa Fluor647-anti-SAM antibody at 8 μg/ml in 200 μl staining buffer (PBS with 1%BSA). Put the plate under 4° C. for 1 h. Add proper amount of DAPI for 5minutes to stain nuclei only.

(7) Washed with 1 ml 1×PBS for 3 times.

(8) Sealed the glass with the special resin that is especially designedto be used for LSCM to prevent fluorescence from being quenched.

(9) Observed and took photos with Zeiss LSM 780 with 630-foldmagnification. The results are shown in FIG. 7 which illustrates theLaser Scan Confocal Microscopy (LSCM) results of L02 and HepG2 cellsthat were cultured for 40 h and then stained with the SAMe fluorescencelabelled anti-SAM and anti-SAH antibodies of the invention. In FIG. 7, Aare the L02 cells stained with anti-SAM antibody; B are the HepG2 cellsstained with anti-SAM antibody; C are L02 cells stained with anti-SAHantibody; and D are HepG2 cells stained with anti-SAH antibody. Thephotos were taken by Zeiss LSM 780 under 630-fold magnification.

C. Fluorescence Immunology in Connection with Streptavidin (SA) andBiotin System

Instead of directly or indirectly labeling FTs to anti-SAM and anti-SAHantibodies, different sizes or colored quantum dots can be labeled ontoSA. (1) SAM and SAH are conjugated with biotin through various linkers(as exemplified in our application Ser. No. 15/091,544 filed Apr. 5,2016, the entire contents of which are incorporated by reference hereinas if they were entirely denoted). (2) Different FTs are labeled ontoSA. (3) Through the specific and strong binding between SA and biotin,small molecule antigen SAM and SAH can be therefore labeled to differentFTs separately, i.e. FT-SAM and FT-SAH are obtained. (4) If wishing tomeasure SAM and SAH simultaneously in a sample, mix the differentcolored FT-SAM and FT-SAH and use competitive mechanism in animmunoassay to quantify SAM and SAH with the use of specific antibodiesagainst SAM and SAH.

D. Fluorescence Immunology in Connection withDigoxingenin—Anti-Digoxingenin Antibody System

Other indirect methods of tracing SAM and SAH include (1) conjugatingSAM or/and SAH to digoxigen or digoxingenin through various linkers (asexemplified in our provisional application Ser. No. 15/091,544 filedApr. 5, 2016, the entire contents of which are incorporated by referenceherein as if they were entirely denoted). (2) Different FTs are labeledonto mouse anti-digoxigenin or mouse anti-digoxin antibodies. (3) Mixproducts from step (1) and step (2), so SAM and SAH are indirectlylabeled to different colored FTs. (4) Uses of FT-SAM and FT-SAH are asdescribed above as in Example 11C.

Example 12—Using the Test Strips to Measure SAM and SAH Levels fromHealthy Human Blood Samples and Monitoring Progress in Weight Reduction

About 5 ml blood samples were drawn via I.V. into heparinized tubes from34 healthy subjects (volunteers from our R&D department, subjects werefasting for at least 5 hours). 100 μl plasma samples were added to theSAM and SAH immunochromatographic test strips as described in theExample 1 and 2 above and the values were read from DryImmunofluorescence Analyzer Model FIC-S2011 series (Arthus Biosystems,VA). As can be seen from the Table 1, the averages of SAM, SAH and MIfrom 15 females were higher than (SAM 25.51%, SAH 74.25%, MI 19.15%higher respectively) those corresponding values from 18 male subjects.

TABLE 1 Levels of SAM, SAH and MI in healthy plasma samples by genderBMI SAM (nM) SAH (nM) MI Gender Case AVG. 22.00 256.80 86.60 5.60 F 15STDEV. 3.93 164.24 37.95 4.45 F 15 AVG. 22.60 204.60 49.70 4.70 M 18STDEV. 2.20 103.85 32.37 2.318 M 18

We further separated groups based on BMI (Body Mass Index) within eachgender group. BMI information was missing for one of the male subjects.The averages of SAM, SAH and MI and the standard deviations were shownin Table 2. The averages of SAM and MI in high BMI (BMI>24) groups wereobviously decreased as compared to those in low BMI (BMI<=24) groups forboth female and male subjects. With average SAM at 143.7 nM from highBMI group versus SAM at 285.07 nM from low BMI group in females, andaverage SAM at 185 nM from high BMI group versus SAM at 214 nM from lowBMI group in males. The average MI from the high BMI group is only30.76% of the MI from the low BMI group in females and about 63.49% inmale subjects. This implied that high BMI had more impacts on (reduced)MIs of females than on males. BMI less than 24 is considered ideal forhealth reason. Therefore, abnormal BMI is related to SAM levels in bothgenders. Low SAM might be the reason for the abnormal and unfavorableBMI that usually underlies a series of health concerns includingcardiovascular and renal diseases, diabetes, obesity and other metabolicdisorders, etc. (Lydi M. J. W. van Driel reported the relationshipbetween BMI and methylation in young females (Body Mass Index Is anImportant Determinant of Methylation Biomarkers in Women of ReproductiveAges, J. Nutr. 139: 2315-2321, 2009.). The results indicated SAM, SAHand MI are good indicators or biomarkers for health issues caused byabnormal BMI, such as cardiovascular diseases.

TABLE 2 Levels of SAM, SAH and MI in healthy plasma samples by genderand BMI BMI >= 24 BMI < 24 Gender Case SAM (nM) SAH (nM) MI Case SAM(nM) SAH (nM) MI AVG. F 3 143.70 85.05 2.0 12 285.06 86.96 6.5 STDEV. F3 88.46 27.42 1.9 12 168.97 102.43 4.4 AVG. M 4 185.00 69.00 4.0 13214.00 48.58 6.3 STDEV. M 4 76.61 52.27 2.1 13 116.06 30.41 4.6

As BMI greater than 24 may indicate unhealthy conditions, we removed 3female and 4 male cases with BMI higher than 24, Table 1 becomes Table 3below. The averages of SAM, SAH and MI from 12 normal BMI females werehigher than (SAM 33.41%, SAH 97.73%, MI 25% higher respectively) thosecorresponding values from 13 male subjects. Furthermore, by removingabnormal BMI cases, the differences of SAM, SAH and MI values betweenfemales and males were even more obvious, i.e. the average female SAMlevel is 33.41% (instead of 25.51% if subjects with all BMI values wereconsidered) higher than that of male if only looking at the normal BMIsubjects in each gender. Table 4 showed abnormal BMI may blur (decrease)the differences in SAM, SAH and MI values between females and males.This indicates BMI is a factor that complicates the values of SAM, SAHand MI, which is consistent with the fact that the levels of SAM and SAHvary according to race, gender, body weight, age and general healthyconditions.

TABLE 3 Levels of SAM, SAH and MI in healthy plasma samples by gender(all BMI < 24) BMI SAM (nM) SAH (nM) MI Gender Case AVG. 20.4 285.1087.00 6.50 F 12 STDEV 2.17 168.97 102.43 4.48 F 12 AVG. 21.70 213.7044.00 5.20 M 13 STDEV 1.60 116.06 25.40 2.41 M 13

TABLE 4 Percent increases of SAM, SAH and MI in females than males SAMSAH MI With all BMI 25.51 74.25 19.15 With healthy BMI (<=24) 33.4197.73 25.00

The correlation between BMI and MI levels and SAM levels appears toindicate that monitoring these biomarkers together with the BMI providepractical information in designing diets for a given set of a patientpopulation.

Example 13—Using the Test Strips to Measure SAH, HCy, CRP and cTnI fromHealthy and Cardiovascular Blood Samples

The abbreviations used in this example are as follows: cTnI (CardiacTroponin-I), CRP, CK-MB (Creatine-kinase-MB) and Myo (Myoglobin),

The 9 healthy and 7 cardiovascular disease (CVD) human blood sampleswere obtained from patients diagnosed as cardio attacks in the clinicallab of The Second Affiliated Hospital of Xiangya Medical School, CentralSouth University in Changsha, Hunan province. The samples were used tomeasure SAM, SAH, MI and CRP using the test strips from the Examples 1,2, 3 and 4 in this invention. The data from Table 5 showed 9 caseswithout acute myocardial injury (AMI) as determined by clinical lab'snegative cTnI, CRP, CK-MB and Myo results had the average SAM value as164 nM, SAH as 232 nM, MI as 1.75 and 44.44% of them with HCy higherthan 15 μM, and CRP values measured using test strip as described in theExample 4 showed an average of 0.8 mg/ml (normal). Whereas, in the 7heart attack cases that were diagnosed with much increased cTnI, CRP,CK-MB and Myo the average SAM value was 94 nM, SAH as 558 nM, MI as 0.2and 85.71% of them with HCy higher than 15 μM. The average CRP for the 7cases with heart attack or AMI is 4.37 mg/l, which was higher thannormal but not related to inflammation reaction as it is less than 10mg/l. For the last two samples with higher Myo (increased in the firstfew hours of AMI), CRP levels were not high and just about to increase.For the first two cases with peak cTnI that normal occurs around 36 hpost-AMI, CRP levels were much elevated. This indicated that CRPelevation happened after about a day or so. The results indicated thatdecreased SAM and MI, increased SAH and HCy are also good biomarkers forheart diseases. Only 1 of the 7 AMI patients showed negative HCy, yetthe SAH level of this patient was extremely higher than other cases,about 5-fold higher than normal average SAH level. This indicated thatSAH, MI are better indicators than HCy in diagnosing heart diseases.

The SAM, SAH, HCy and CRP values measured for all samples using theimmunochromatographic test strips described in the Example 4 in thisinvention may help identify and sort out certain groups of patients thatmay be overlooked by merely checking cTnI, CK-MB, Myo and HCy alone withphotochemical methods that are currently often checked. SAM, SAH, HCyand CRP are useful biomarkers that will add to the current cardiac panelin order to timely diagnose, differentiate, predict the prognosis andhelp direct treatment of CVDs.

TABLE 5 Measurement of biomarkers in clinical samples (n = 16) MarkerSAM CRP (ng/ml) Value CVD (nM) SAH (nM) MI (mg/l) HCy cTnI 0.035 − 36740 9.12 0.30 − cTnI 0.22 − 48 50 0.95 0.31 + cTnI 0.239 − 666 207 3.210.58 − CRP 1.26 − 35 129 0.27 1.21 + CRP 1.51 − 83 522 0.16 1.49 − CK-MB18.92 − 26 303 0.09 1.14 − Myo 54.98 − 48 273 0.18 0.77 − Myo 33.93 − 45466 0.10 0.48 + Myo 67.88 − 160 95 1.68 0.92 + AVG. 164 232 1.75 0.80STDEV. 217 175 2.95 0.42 cTnI >50 + 72 405 0.18 5.75 + cTnI 45.6 + 2111320 0.16 5.21 − CRP 7.73 + 33 521 0.06 7.02 + CK-MB 54.95 + 48 98 0.494.51 + CK-MB 33.93 + 85 456 0.19 3.33 + Myo 119.39 + 138 610 0.23 1.24 +Myo 152.47 + 73 709 0.10 3.52 + AVG 94 588 0.20 4.37 STDEV. 61 376 0.141.88

Based on experiments that have been conducted, as set forth in some ofthe preceding examples, it is believed that quantum dot probes andfluorescent chelates provide higher fluorescence than that provided byother probes that have been labeled with organic fluorescent molecules;and their longer lasting fluorescence allow for stable and reliablesystems to be built; Therefore, it is believed that quantum dot andfluorescent chelate based assays for determining SAM, SAH and HCyprovide series of advantages over assays that employ traditional organicfluorescent molecules.

The entire contents of the following provisional and non-provisionalapplications are incorporated by reference into the presentnon-provisional application as if they were denoted in their entirety:

U.S. Ser. No. 14/457,099 filed Aug. 11, 2014;

U.S. Ser. No. 14/218,928 filed Mar. 18, 2014;

U.S. Provisional Patent Application No. 61/801,547 filed on Mar. 15,2013; and

U.S. Provisional Patent Application No. 62/143,790 filed Apr. 6, 2015.

Although the foregoing description (Angres) contains many specifics,these should not be construed as limiting the scope of the presentinvention, but merely as providing illustrations of some of thepresently preferred embodiments. Similarly, other embodiments may bedevised without departing from the spirit or scope of the presentinvention. Features from different embodiments may be employed incombination. The scope of the invention is, therefore, indicated andlimited only by the appended claims and their legal equivalents ratherthan by the foregoing description. All additions, deletions andmodifications to the invention as disclosed herein which fall within themeaning and scope of the claims are to be embraced thereby.

What is claimed is:
 1. A fluorescent material conjugated to a monoclonalanti-SAM antibodies: wherein said fluorescent material is a fluorescentlanthanide chelate and wherein said monoclonal anti-SAM antibodies arederived from hybridomas having the designation from the China Center ForType Culture Collection (CCTCC) number C2017178 and number C2017179. 2.The conjugate of claim 1, wherein said lanthanide is europium andterbium.
 3. An immunochromatographic strip having incorporated thereinfluorescent material of claim 1.